An analytical method suited for biological samples is indispensable for the determination of human exposure to TCC. The first aim of our study was to develop a rapid, accurate and robust analytical approach for the parallel quantification of TCC, its analogs and metabolites in biological fluids. Through the application of online-SPE sample preparation prior to LC-MS/MS analysis, sample preparation was minimized. Overall, only four steps required manual labour, (i) sample collection (ii) mixing with I.S. solution (iii) centrifugation and (iv) transferring the supernatant to the vial. Considering that 30 samples are centrifuged at the same time, cumulative hands on preparation time required per sample is less than 1 min per sample. The online SPE-LC-ESI-MS/MS approach developed herein is not only rapid (7 min/sample), but has excellent accuracy and precision for urine and plasma samples. Due to its sensitivity, it is possible to accurately monitor TCC and concurrently its major oxidative metabolites in these biological samples without the need to expose human subjects to radioactivity as described earlier14, 15
Following showering with a soap containing 0.6% TCC, the renal excretion profile obtained, including the presence of TCC-N-Gs as major metabolites and the lack of oxidative metabolites, is consistent with all previous studies on the human metabolism of TCC15, 20, 42
. . The levels of TCC-N-G () are consistently higher than those reported from previous showering or bathing studies with TCC containing soaps. Specifically, Gruenke et al. reported 30 ng/mL TCC-N-Gs (60 nM) in the urine of users of TCC containing bar soaps, though the TCC content of these soaps and bathing procedures were not reported42
. Interestingly, Howes and Black did not find TCC (<25 ng/mL) in the urine of human subjects after intensive 28-day bathing with 2% TCC containing soap16
. However, this is most likely due to a lack of appropriate conjugate hydrolysis.
In contrast to earlier human exposure studies, here the amounts of excreted TCC in urine were quantified in each urination following exposure. Therefore we demonstrated for the first time that renal excretion of TCC varies widely among individual subjects (). The maximal TCC-N-Gs concentrations in the urine were detected 12–24h after exposure with up to a 10-fold variation among individuals (table S4
). These differences did not correlate with age, height or weight BMI and body surface area of the volunteers (Table S3
) and are most likely due to individual differences in absorption, distribution, metabolism and excretion. The total clearance of TCC in urine following a single exposure required approximately 72 hr. In order to investigate if TCC accumulated due to its slow excretion, we quantified the urine concentrations of TCC and its metabolites over a time period of two weeks of daily showering with TCC in a single individual. No accumulation of TCC or its metabolites was detected (), but urinary excretion reached a constant level of 79±20 μg TCC-N-Gs g−1
creatinine indicating a steady state of TCC body burden.
The large amount of TCC extracted as TCC-N-Gs via the urine in the subjects demonstrates that a relevant portion of TCC (70±15 mg, equivalent to a topical dose 1 mg/kg BW) was absorbed after showering. Considering a renal excretion rate of 25%14, 15
and a constant creatinine excretion rate of 1.5 g/24 h, we estimated that the mean absorption of TCC was 0.5±0.1 mg corresponding to 0.6±0.2% of the dose applied (a detailed description of the model used is given in SI
). This result is consistent with previous findings of 0.4% absorption of the TCC applied in a similar showering study using 14
C labelled TCC14
. This good correlation argues that the TCC-N-Gs levels in urine are highly predictive of the human TCC exposure and TCC-N-Gs levels are ideally suited for monitoring TCC exposure.
Our in vitro
screening results indicate that TCC does not affect the activity of human carboxylesterases, fatty acid amidase, microsomal epoxide hydrolases, cytochrome P450 and glutathione S-transferases. However we could show with two independent methods that TCC is a nanomolar inhibitor of sEH. TCC exhibits a similar inhibitory potency (IC50
) when recombinant human and rat enzymes are compared (). Thus, the rat may be a suitable model for humans when investigating the biological effects of TCC mediated by sEH inhibition. Since the preliminary rat exposure study described here did not lead to systemic inhibition of sEH as measured by oxylipin profiles (Fig. S13
), it seems possible that the systemic concentrations of free TCC (non-conjugated) found in humans through showering may not be sufficient to cause effects through sEH inhibition. However at the site of topical application, the TCC concentration might reach levels which lead to local effects via sEH inhibition. Possible reasons for the lack of systemic effects of TCC could be non-covalent protein binding, causing a low uptake into the cells. A key finding of this study is the deactivation of the inhibitory potency of TCC on sEH by its metabolism. As demonstrated in this study all investigated TCC metabolites show a lower potency than the parent compound (). The in vitro
potency of TCC-N-Gs was not determined since reference compounds were not available. However, based on the extensive SAR of sEH inhibitors it is highly unlikely that these major urinary metabolites would inhibit sEH43–45
. We predict that the glucuronic acid group would prevent hydrogen binding of the urea moiety in the active site of the enzyme, and based on our crystal structure data TCC-N-G′s are too large to fit in the catalytic pocket.
Overall our human exposure study in a small group of subjectsshowed that a portion of the TCC present in bar soaps is absorbed through the skin and is excreted in urine as N-glucuronides. These urinary glucuronides appear to be valuable biomarkers of TCC exposure.. TCC is a nanomolar inhibitor of sEH, and inhibition of this enzyme has been shown to have profound though largely beneficial effects on mammalian physiology. Our studies of TCC exposure in human volunteers using a commercial soap when compared to preliminary exposure and efficacy data in the rat suggest that such TCC exposures are unlikely to elicit systemic changes based on sEH inhibition. However, local dermal effects cannot be ruled out. The TCC exposures found following showering also indicate that a careful risk benefit analysis of TCC in personal care products should be undertaken. In particular, long term exposure studies in humans that include bio-monitoring of TCC in blood should be carried out to evaluate if exposure after using PCPs containing up to 1.5% TCC provide a sufficient margin of safety. With the online-SPE-LC-MS/MS method described herein, we provide an excellent analytical tool to answer these questions.